A complex of anti-hyaluronidase substances has been derived from several natural products, such as small fish containing amino acids, whole glucides, glycogen chondroitinsulphate, phenol, hypotensive substances, with an antihyaluronidase activity of a minimum of 50 I.U/ml.
The method whereby the complex is produced consists of a two-phased treatment of se fish with 0.5% phenol solution, over a period of 24 h, the extracts brought together are vacuum concentrated at a maximum 40.degree. C., then they are treated with an equal ethanol 96.sup.c volume; the precipitate is removed and the supernatant obtained is treated with celite, filtered, vacuum-concentrated at a maximum 40.degree. C., then solvent-extracted--treated and after removing the solvent the supernatant is treated with phenol up to a value of 4.5-5.5%.
Drug compositions containing an anti-hyaluronidase bioactive extract, phenol and sodium metabisulphite, have been developed too.
These drugs rely on a complex of substances, natural active principles, associated in proportions close to the normal, functional ones, produced by extraction from animal tissues or biosynthesized in cells and microorganism cultures, the role of this complex being to replace, to substitute for or even to allow recovery of the naturally existing complex in connective tissues, that had been unbalanced for various reasons and which caused the disease.
The extraction and conditioning processes have been established so that losses and degradation of active substances should be as low as possible and when the ratio of raw material constituents is different from that one required to treat disease in human being, it may be set off by a proper compounding of extracts or by a treatment schedule.
Now, the disease-inducing mechanisms are known to result from a disequilibrium in the biosynthesis and breakdown of normal constituents on which the proper movement of joints depends.
Cells in the tissues of the living body are embedded and evolve within basic substances. This basic substance--a general compound of living structures-pervades every interspace and isolates every stationary cell from its neighbors. Variations in the composition of the extracellular environment exert a profound influence on cell behavior and in turn the cells possess a powerful means of modifying their immediate environment.
The intercellular substance is a complex gel containing water, electrolytes, metabolites, dissolved gases, enzymes, trace elements, fats, proteins, carbohydrates. This substance is rendered highly viscous by an abundance of certain long-chain acid mucopolysaccharide polymers, particularly glycosaminoglycans and the related proteoglycans, reinforced at the microscopic level by a three-dimensional network of collagen fibrils (Cameron, E. and Pauling, L--The Encyclopedia of Ignorance, p. 377-385, Pergamon Press, Oxford, 1982).
The role of synovial fluid, found in all joints, is not only to lubricate the moving structures but also to dissipate the energy.
This function is performed by the synovial fluid composition where the glycosaminoglycans (GAC) and proteoglycans (PG such as hyaluronic acid and chondroitinsulphate inducing a high viscosity with their spiral structure, behave viscous-like when the moving frequencies are low and elastic--like when the moving frequencies are high (Ogston, A. G. and Stanier, J. E. (1953)--J. Physiol, 199, 124).
Elasticity is due to the presence of the highly concentrated hyaluronic acid, at a pH=2.5 which forms a viscous-elastic paste in a salt solution (Balasz, E. A., Chemistry and Molecular Biology of the Intercellular Matrix, Vol. II and III, Academic Press, New York, 1970).
At different ages, the synovial fluid behaves similarly as it concerns viscosity, but the energy taking over is different at high moving frequencies, the synovial fluid in young people taking over 77% of the energy while in the old it takes over only 52% of the energy by elastic storage.
The synovial fluid in affected joints (osteoarthrosis) is viscous, but not elastic. The stiffness of this synovial fluid is 7 times lower than that one in the healthy old people. The hyaluronic acid concentration in these fluids is low, resulting in low viscosity indices and the protein concentration is high. The negative influence of concentration change may lower in some cases, the viscosity index up to 30 times.
Also, in joints, tendons, vitreous bodies of the eye--the fluid area (synovial fluid, vitreous fluid) is adjacent to the solid matrix (articular cartilage, tendon, fascia, vitreous body).
The viscous-elastic macromolecular compound--the hyaluronic acid--in the fluid matrix, penetrate the solid matrix surface. Rheologically, the major difference between the two matrices is the presence of collagen fibrils in the solid matrix and their absence in the fluid one.
The surface layer of 1-2.mu. consists of hyaluronic acid and proteins. This layer can be removed or destroyed by the action of hyaluronidase, found in excess in the intercellular matrix.
The sublayer of 10-15.mu. in thickness contains typical collagen fibrils, and the interfibrillar space is filled with approximately equal amount of hyaluronic acid and chondroitinsulphate. on the basis of the data known from the specialized field (Cameron, E. and Pauling, L--The Encyclopedia of Ignorance, p. 377-385, Pergamon Press, Oxford, 1982 and Balasz, E. A. and Sweeney, D. B.--11968--New and Controversial Aspects of Retinal Detachment, p. 371, Ed. McPerson, Harpen and Row, N.Y.), this invention aimed at producing a complex of ingredients in which the mucopolysaccharide polymers and proteoglycans take part under the form of hyaluronic acid and chondroitinsulphate, as well as their fragments, herparin and ascorbic acid, all of them being substances certified by recent investigations, contributing to restore the normal functions in the connective tissues, in the case of degenerative diseases. These diseases, altering the basic substance in which the cells are embedded, deteriorates the relationship existing between the basic substance and these cells. The condition for a "normal" balance is for each part--the basic substance and the cell--to keep its own functions, since any change, as for instance the reduction in basic substance viscosity by hyaluronic acid depolymerizing through the action of hyaluronidase secreted in excess by cells, is followed by disease occurrence.
Another alternative to restore the balance is the association of an antienzyme called anti-hyaluronidase (hyaluronidase is after all, an enzyme group with the same functions as the connective structures), that stops or limits the hyaluronic acid depolymerization, alongside chondroitinsulphate, heparin and ascorbic acid.
Some attempts have been already known, to prepare drugs based on anti-hyaluronidase which is extracted from its containing sources, such as: bovine trachea, bovine and sheep testicles, umbilical cord, small sea fish, etc.
Although important at that time (Patent RO/82273-1982 and Technological Reports drawn up by the Institute of Chemical and Pharmaceutical Research-Bucharest, No. 84740/1977 and No. 5160/1979), these attempts represented an elementary alternative solving only partially the question of both producing method and the composition of the drugs achieved.